ABSTRACT Tuberculosis (TB) is the deadliest global infection (1.4 million deaths in 2015), having recently surpassed HIV/AIDS. ~250,000 of these deaths were from multidrug-resistant (MDR) TB, with only ~50% success in treatment of MDR-TB. Unless new strategies to combat or prevent emergence of drug-resistant TB are found, the global spread of MDR-TB will be an epidemic of epic proportions. Kanamycin A (KAN) is an antibiotic of last resort used to treat MDR-TB. Resistance to KAN signifies extensively drug-resistant (XDR) TB, which is nearly incurable. One-third of KAN-resistant TB is caused by upregulation of the acetyltransferase Eis in Mycobacterium tuberculosis (Mtb), caused by mutations in the eis promoter. We investigated the mechanism of Eis and discovered and validated several Eis inhibitor scaffolds in the previous funding period. In Aims 1 and 2 of this application, we propose to develop a novel strategy to combat and forestall KAN resistance in TB. These Aims are focused on the preclinical development of these Eis inhibitors for their ultimate use as a combination therapy with KAN against MDR-TB. In Aim 3, we propose to determine the mechanism(s) of the anti-Mtb activity of novel compounds that were discovered to inhibit Eis and potently inhibit Mtb bacteria without KAN, ultimately to develop them as therapeutics against both drug-resistant and drug-resistant TB.